Isolated nona- and decapeptides which bind to HLA molecules, and the use thereof

ABSTRACT

Nonapeptides and decapeptides which bind to HLA molecules and provoke proliferation of cytolytic T cells are disclosed. The decapeptides terminate in Valine, and are restricted in their first three amino acid positions. Other useful nonapeptides are also disclosed.

RELATED APPLICATION

This is a continuation-in-part of Ser. No. 08/880,963, filed Jun. 23,1997, now U.S. Pat. No. 6,025,470 incorporated by reference. A portionof the invention was published by the inventors, less than one yearbefore the filing date of this continuation-in-part application. SeeRomero et al., J. Immunol. 159: 2366 (1997) incorporated by reference.

FIELD OF THE INVENTION

This invention relates to peptides which are useful in the context ofcellular immunology. More particularly, the invention relates topeptides which bind to HLA molecules on the surface of cells. At leastsome of these peptides also induce the activation of cytolytic T cells,when they are complexed with their partner HLA molecule. Also a part ofthe invention are the uses of these peptides in areas such asidentifying HLA-A2 positive cells, provoking T cells, determiningpresence of particular T cells, as well as cytolytic T cells themselves.

BACKGROUND AND PRIOR ART

The study of the recognition or lack of recognition of cancer cells by ahost organism has proceeded in many different directions. Understandingof the field presumes some understanding of both basic immunology andoncology.

Early research on mouse tumors revealed that these displayed moleculeswhich led to rejection of tumor cells when transplanted into syngeneicanimals. These molecules are “recognized” by T-cells in the recipientanimal, and provoke a cytolytic T-cell response with lysis of thetransplanted cells. This evidence was first obtained with tumors inducedin vitro by chemical carcinogens, such as methylcholanthrene. Theantigens expressed by the tumors and which elicited the T-cell responsewere found to be different for each tumor. See Prehn, et al., J. Natl.Canc. Inst. 18: 769-778 (1957); Klein et al., Cancer Res. 20: 1561-1572(1960);

Gross, Cancer Res. 3: 326-333 (1943), Basombrio, Cancer Res. 30:2458-2462 (1970) for general teachings on inducing tumors with chemicalcarcinogens and differences in cell surface antigens. This class ofantigens has come to be known as “tumor specific transplantationantigens” or “TSTAs”. Following the observation of the presentation ofsuch antigens when induced by chemical carcinogens, similar results wereobtained when tumors were induced in vitro via ultraviolet radiation.See Kripke, J. Natl. Canc. Inst. 53: 333-1336 (1974).

While T-cell mediated immune responses were observed for the types oftumor described supra, spontaneous tumors were thought to be generallynon-immunogenic. These were therefore believed not to present antigenswhich provoked a response to the tumor in the tumor carrying subject.See Hewitt, et al., Brit. J. Cancer 33: 241-259 (1976). The family oftum⁻ antigen presenting cell lines are immunogenic variants obtained bymutagenesis of mouse tumor cells or cell lines, as described by Boon etal., J. Exp. Med. 152: 1184-1193 (1980), the disclosure of which isincorporated by reference. To elaborate, tum⁻ antigens are obtained bymutating tumor cells which do not generate an immune response insyngeneic mice and will form tumors (i.e., “tum⁺” cells). When thesetum⁺ cells are mutagenized, they are rejected by syngeneic mice, andfail to form tumors (thus “tum⁻”). See Boon et al., Proc. Natl. Acad.Sci. USA 74: 272 (1977), the disclosure of which is incorporated byreference. Many tumor types have been shown to exhibit this phenomenon.See, e.g., Frost et al., Cancer Res. 43: 125 (1983).

It appears that tum⁻ variants fail to form progressive tumors becausethey initiate an immune rejection process. The evidence in favor of thishypothesis includes the ability of “tum⁻” variants of tumors, i.e.,those which do not normally form tumors, to do so in mice with immunesystems suppressed by sublethal irradiation, Van Pel et al., Proc. Natl.Acad. Sci. USA 76: 5282-5285 (1979); and the observation thatintraperitoneally injected tum⁻ cells of mastocytoma P815 multiplyexponentially for 12-15 days, and then are eliminated in only a few daysin the midst of an influx of lymphocytes and macrophages (Uyttenhove etal., J. Exp. Med. 152: 1175-1183 (1980)). Further evidence includes theobservation that mice acquire an immune memory which permits them toresist subsequent challenge to the same tum⁻ variant, even whenimmunosuppressive amounts of radiation are administered with thefollowing challenge of cells (Boon et al., Proc. Natl, Acad. Sci. USA74: 272-275 (1977); Van Pel et al., supra; Uyttenhove et al., supra).Later research found that when spontaneous tumors were subjected tomutagenesis, immunogenic variants were produced which did generate aresponse. Indeed, these variants were able to elicit an immuneprotective response against the original tumor. See Van Pel et al., J.Exp. Med. 157: 1992-2001 (1983). Thus, it has been shown that it ispossible to elicit presentation of a so-called “tumor rejection antigen”in a tumor which is a target for a syngeneic rejection response. Similarresults have been obtained when foreign genes have been transfected intospontaneous tumors. See Fearon et al., Cancer Res. 48: 2975-1980 (1988)in this regard.

A class of antigens has been recognized which are presented on thesurface of tumor cells and are recognized by cytolytic T cells, leadingto lysis. This class of antigens will be referred to as “tumor rejectionantigens” or “TRAs” hereafter. TRAs may or may not elicit antibodyresponses. The extent to which these antigens have been studied, hasbeen via cytolytic T cell characterization studies, in vitro i.e., thestudy of the identification of the antigen by a particular cytolytic Tcell (“CTL” hereafter) subset. The subset proliferates upon recognitionof the presented tumor rejection antigen, and the cells presenting thetumor rejection antigens are lysed. Characterization studies haveidentified CTL clones which specifically lyse cells expressing the tumorrejection antigens. Examples of this work may be found in Levy et al.,Adv. Cancer Res. 24: 1-59 (1977); Boon et al., J. Exp. Med. 152:1184-1193 (1980); Brunner et al., J. Immunol. 124: 1627-1634 (1980);Maryanski et al., Eur. J. Immunol. 124: 1627-1634 (1980); Maryanski etal., Eur. J. Immunol. 12: 406-412 (1982); Palladino et al., Canc. Res.47: 5074-5079 (1987). This type of analysis is required for other typesof antigens recognized by CTLs, including minor histocompatibilityantigens, the male specific H—Y antigens, and the class of antigensreferred to as “tum-” antigens, and discussed herein.

A tumor exemplary of the subject matter described supra is known asP815. See DePlaen et al., Proc. Natl. Acad. Sci. USA 85: 2274-2278(1988); Szikora et al., EMBO J 9: 1041-1050 (1990), and Sibille et al.,J. Exp. Med. 172: 35-45 (1990), the disclosures of which areincorporated by reference. The P815 tumor is a mastocytoma, induced in aDBA/2 mouse with methylcholanthrene and cultured as both an in vitrotumor and a cell line. The P815 line has generated many tum⁻ variantsfollowing mutagenesis, including variants referred to as P91A (DePlaen,supra), 35B (Szikora, supra), and P198 (Sibille, supra). In contrast totumor rejection antigens—and this is a key distinction—the turn antigensare only present after the tumor cells are mutagenized. Tumor rejectionantigens are present on cells of a given tumor without mutagenesis.Hence, with reference to the literature, a cell line can be tum⁺, suchas the line referred to as “P1”, and can be provoked to producetum⁻variants. Since the tum⁻ phenotype differs from that of the parentcell line, one expects a difference in the DNA of tum⁻ cell lines ascompared to their tum⁺ parental lines, and this difference can beexploited to locate the gene of interest in tum⁻ cells. As a result, itwas found that genes of tum⁻ variants such as P91A, 35B and P198 differfrom their normal alleles by point mutations in the coding regions ofthe gene. See Szikora and Sibille, supra, and Lurquin et al., Cell 58:293-303 (1989). This has proven not to be the case with the TRAs of thisinvention. These papers also demonstrated that peptides derived from thetum⁻ antigen are presented by H-2^(d) Class I molecules for recognitionby CTLs. P91A is presented by L^(d), P35 by D^(d) and P198 by K^(d).

PCT application PCT/US92/04354, filed on May 22, 1992 assigned to thesame assignee as the subject application, teaches a family of humantumor rejection antigen precursor coding genes, referred to as the MAGEfamily. Several of these genes are also discussed in van der Bruggen etal., Science 254: 1643 (1991). It is now clear that the various genes ofthe MAGE family are expressed in tumor cells, and can serve as markersfor the diagnosis of such tumors, as well as for other purposesdiscussed therein. See also Traversari et al., Immunogenetics 35: 145(1992); van der Bruggen et al., Science 254: 1643 (1991) and De Plaen,et al., Immunogenetics 40: 360 (1994). The mechanism by which a proteinis processed and presented on a cell surface has now been fairly welldocumented. A cursory review of the development of the field may befound in Barinaga, “Getting Some ‘Backbone’: How MHC Binds Peptides”,Science 257: 880 (1992); also, see Fremont et al., Science 257: 919(1992); Matsumura et al., Science 257: 927 (1992); Engelhard, Ann. Rev.Immunol 12:181-207 (1994); Madden, et al, Cell 75:693-708 (1993);Ramensee, et al, Ann. Rev. Immunol 11:213-244 (1993); Germain, Cell 76:287-299 (1994). These papers generally point to a requirement that thepeptide which binds to an MHC/HLA molecule be nine amino acids long (a“nonapeptide”), and to the importance of the second and ninth residuesof the nonapeptide. For H-2K^(b), the anchor residues are positions 5and 8 of an octamer, for H-2D^(b), they are positions 5 and 9 of anonapeptide while the anchor residues for HLA-A1 are positions 3 and 9of a nonamer. Generally, for HLA molecules, positions 2 and 9 areanchors.

Studies on the MAGE family of genes have now revealed that a particularnonapeptide is in fact presented on the surface of some tumor cells, andthat the presentation of the nonapeptide requires that the presentingmolecule be HLA-A1. Complexes of the MAGE-1 tumor rejection antigen (the“TRA” or nonapeptide”) leads to lysis of the cell presenting it bycytolytic T cells (“CTLs”).

Research presented in, e.g., U.S. Pat. No. 5,405,940 filed Aug. 31,1992, and in U.S. Pat. No. 5,571,711, found that when comparinghomologous regions of various MAGE genes to the region of the MAGE-1gene coding for the relevant nonapeptide, there is a great deal ofhomology. Indeed, these observations lead to one of the aspects of theinvention disclosed and claimed therein, which is a family ofnonapeptides all of which have the same N-terminal and C-terminal aminoacids. These nonapeptides were described as being useful for variouspurposes which includes their use as immunogens, either alone or coupledto carrier peptides. Nonapeptides are of sufficient size to constitutean antigenic epitope, and the antibodies generated thereto weredescribed as being useful for identifying the nonapeptide, either as itexists alone, or as part of a larger polypeptide.

The preceding survey of the relevant literature shows that variouspeptides, usually eight, nine, or ten amino acids in length, complexwith MHC molecules and present targets for recognition by cytolytic Tcells. A great deal of study has been carried out on melanoma, andmelanoma antigens which are recognized by cytolytic T cells are nowdivided into three broad categories. The first, which includes many ofthe antigens discussed, supra, (e.g., MAGE), are expressed in somemelanomas, as well as other tumor types, and normal testis and placenta.The antigens are the expression product of normal genes which areusually silent in normal tissues.

A second family of melanoma antigens includes antigens which are derivedfrom mutant forms of normal proteins. Examples of this family are MUM-1(Coulie, et al, Proc. Natl. Acad. Sci. USA 92:7976-7980 (1955)); CDK4(Wolfel, et al, Science 269:1281-1284(1955)); B. Catenin (Robbins, etal, J. Exp. Med. 183:1185-1192 (1996)); and HLA-A2 (Brandel, et al, J.Exp. Med. 183:2501-2508 (1996)). A third category, also discussed,supra, includes the differentiation antigens which are expressed by bothmelanoma and melanocytes. Exemplary are tyrosinase, gp100, gp75, andMelan A/Mart-1. See U.S. Pat. No. 5,620,886 incorporated by reference,with respect to Melan-A. See Wolfel, et al., Eur. J. Immunol. 24: 759(1994) and Brichard, et al., Eur. J. Immunol. 26: 224 (1996) fortyrosinase; Kang, et al., J. Immunol. 155: 1343 (1995); Cox, et al.,Science 264: 716 (1994); Kawakami, et al., J.

Immunol. 154: 3961 (1995) for gp 100; Wang, et al., J. Exp. Med. 183:1131 (1996) for gp 75.

Cytolytic T cells (“CTLs” hereafter) have been identified in peripheralblood lymphocytes, and tumor infiltrating lymphocytes, of melanomapatients who are HLA-A*0201 positive. See Kawakami, et al, Proc. Natl.Acad. Sci, USA 91:3515 (1994); Coulie, et al, J. Exp. Med. 180:35(1994). When ten HLA-A*0201 restricted Melan-A specific CTLs derivedfrom different patients were tested, nine of them were found torecognize and react with the peptide Ala Ala Gly Ile Gly Ile Leu ThrVal, (SEQ ID NO: 2), which consists of amino acids 27-35 of Melan-A.(Kawakami, et al, J. Exp. Med 180:347-352 (1994)). Rivoltini, et al, J.Immunol 154:2257 (1995), showed that Melan-A specific CTLs could beinduced by stimulating PBLs from HLA-A*0201 positive normal donors, andmelanoma patients, using SEQ ID NO: 2. The strength of this response hasled to SEQ ID NO: 2 being proposed as a target for vaccine development.It has now been found, however, that a decapeptide, i.e.,

Glu Ala Ala Gly Ile Gly Ile Leu Thr Val

(SEQ ID NO: 1), is actually a better target than SEQ ID NO: 2. Thisrecognition has led to work set forth herein, which is part of theinvention.

The majority of peptides which have been identified as binding toHLA-A*0201 are 9 or 10 amino acids in length, and are characterized bytwo anchor residues. The first is Leu or Met at position 2, and thesecond is Leu or Val at position 9. See Falk, et al, Nature 351:290(1991). Ruppert, et al, in Cell 74:929 (1993), show that amino acidsfound at other positions within a nona-or decapeptide may also have arole in the peptide -HLA-A*0201 interaction. They show, e.g., that anegatively charged residue or proline at position 1 was associated withpoor HLA-A*0201 binding.

What is interesting about this work is that the two peptides representedby SEQ ID NOS: 1 and 2 do not possess the major anchor residue atposition 2 and, the strong binder SEQ ID NO: 1 has a negatively chargedresidue at position 1.

A strong binder is not necessarily a stable binder, meaning that theinteraction between peptide and HLA molecule may be, and is, brief. Whenit is desired to induce CTLs, to identify them or to carry out othertypes of experiments, it would be desirable to have a peptide with theability to bind to an MHC Class I molecule which binds with highaffinity and forms stable complexes.

The invention involves, inter alia, the development of new nonamers anddecamers which are surprisingly good HLA binders and CTL stimulators.These molecules, as well as their uses, are among the features of theinvention which are set forth in the disclosure which follows.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1(a and b) shows results of experiments carried out to determineif tumor infiltrating lymphocyte populations would lyse cells presentingcomplexes of HLA-A*0201 and various peptides on their surfaces.

FIGS. 2a-2 d show stability studies comparing various peptides.

FIG. 3 shows antigenic activity of various peptides when tested withTILNs.

FIG. 4 parallels FIG. 3, but uses CTLs generated from PBLs bystimulation with various peptides (SEQ ID NO: 11 and SEQ ID NO: 1).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1

In these experiments, tumor infiltrating lymphocytes (“TILs” hereafter),were generated from tumor invaded lymph nodes of patients who wereHLA-A*0201 positive. The experiments were designed so as to avoidantigen specific selection in vitro, and the methodology is now setforth.

Biopsies of tumor infiltrated lymph nodes (“TILNs” hereafter) were lysedto single cell suspensions, and then cultured in 24 well culture plates.A total of 50 μl of cell suspension was added to 2 mls of Iscove'sDulbecco medium which had been supplemented with Asn (0.24 mM), Arg(0.55 mM), and Gln (1.5 mM), and 10% pooled human A⁺ serum (serumobtained from type A blood donors), together with recombinant human IL-2(100 u/ml), and IL-7 (10 ng/ml). These were the only cytokines used toculture the cell suspensions, so as to avoid antigen specific selectionin vitro. The suspensions were cultured for 2-3 weeks, and the cellswere then characterized for cell surface phenotype. Only populationswith greater than 75% CD8⁺T cells, and of desired cytolytic activitywere used. This second property was determined by combining the TILNpopulations with autologous cells, a melanoma cell line previouslycharacterized as being HLA-A*0201 positive (Me290), a melanoma cell lineknown to be HLA-A*0201 negative (Me260) or cell line T2, which does notprocess antigen, together with the peptide of SEQ ID NO: 1. The peptidewas added at 1 μM, together with varying ratios of effector (TILN)cells, and the target cells. The results presented in FIG. 1, showresults obtained using LAU 132 and LAU 203, two TILN populationsidentified via this method. In FIG. 1, “M10” is SEQ ID NO: 1, and theadditional abbreviations are as set forth, supra. The assay was a 4 hour⁵¹Cr release assay, carried out in the absence or presence ofexogenously added peptide. In FIG. 1, open symbols stand for the absenceof the peptide, and solid symbols for its presence. In this assay, thetarget cells were labelled with ⁵¹Cr for one hour, at 37° C., and werethen washed two times. Labelled cells (1000 cells in 50 μl) were addedto a 50 μl sample of effector cells (varying amounts, as indicatedherein), in the presence or absence of 50 μl of antigenic peptide (1μg/ml). Prior to their addition, the effector cells had been incubatedfor a minimum of 20 minutes at 37° C., in the presence of unlabellednatural killer (NK) cell targets (50,000 cells per well), in order toeliminate any non-specific lysis due to NK-like effectors present in theeffector population. The ⁵¹Cr release was measured after 4 hours ofincubation at 37° C., and percent specific lysis was calculated as:$\frac{100\lbrack ( {{{experimental}\quad {release}} - {{spontaneous}\quad {release}}} ) \rbrack}{{total} - {{spontaneous}\quad {release}}}$

As FIG. 1 shows, the two TILN populations lysed the HLA-A*0201 positivecell line equally well, whether or not the peptide was added. TheHLA-A*0201 negative line, Me260, was not lysed in either situation, andT2, which does not process antigen, was lysed only when the peptide wasadded. These results show that the two TILN populations used hereafterrecognize the epitope defined by SEQ ID NO: 1, when complexed toHLA-A*0201 positive cells.

EXAMPLE 2

The experiments described, supra, were modified somewhat, to determineif the TILNs recognized other peptides better than SEQ ID NO: 1. Inthese experiments, the following peptides were synthesized, using knownmethods

Ala Ala Gly Ile Gly Ile Leu Thr Val (SEQ ID NO: 2)

Ala Ala Gly Ile Gly Ile Leu Thr Val Ile (SEQ ID NO: 3)

Ile Leu Thr Val Ile Leu Gly Val Leu (SEQ ID NO: 4)

These peptides correspond, respectively, to amino acids 27-35, 27-36,and 32-40 of Melan-A.

TILN recognition was determined by incubating T2 cells (target), withTILNs (effector), at an effector: target ratio of 30:1. Varyingconcentrations of the peptides SEQ ID NOS: 1, 2, 3 or 4 were used. The⁵¹Cr release assay discussed, supra, was used. The following Table setsforth the results of these experiments, wherein the peptideconcentration is that which gave 50% of maximum activity. Relativeactivity is that obtained via comparison to SEQ ID NO: 2, i.e.:[nM]50%[SEQ ID NO:2][nM50%][test peptide].

TABLE I TILN LAU 203 TILN LAU 132 Peptide^(a) Relative Peptide RelativePeptide Sequence [nM] 50% activity^(b) [nM] 50% activity AAGIGILTV₂₇₋₃₅40 1 15 1 (SEQ ID NO: 2) EAAGIGILTV₂₆₋₃₅ 1.5 27 1 15 (SEQ ID NO: 1)AAGIGILTVI₂₇₋₃₆ 600 0.06 300 0.05 (SEQ ID NO: 3) ILTVILGV₃₂₋₄₀ >10⁴ <4 ×10⁻³ >10⁴ <1.5 × 10⁻³

It will be seen that SEQ ID NO: 1 had significantly higher activity thanthe other peptides tested.

EXAMPLE 3

A series of peptides were then synthesized, in order to attempt todetermine peptides with enhanced binding to HLA-A*0201 molecules. Thepeptides synthesized are considered to be derivatives of SEQ ID NO: 2(i.e., Ala Ala Gly Ile Gly Ile Leu Thr Val), and are

Ala Leu Gly Ile Gly Ile Leu Thr Val (SEQ ID NO: 5)

Ala Met Gly Ile Gly Ile Leu Thr Val (SEQ ID NO: 6)

Leu Ala Gly Ile Gly Ile Leu Thr Val (SEQ ID NO: 7) and

Met Ala Gly Ile Gly Ile Leu Thr Val (SEQ ID NO: 8)

For SEQ ID NO: 1, i.e., Glu Ala Ala Gly Ile Gly lie Leu Thr Val thederivatives were:

Glu Leu Ala Gly Ile Gly Ile Leu Thr Val (SEQ ID NO: 9)

Glu Met Ala Gly Ile Gly Ile Leu Thr Val (SEQ ID NO: 10)

Glu Ala Leu Gly Ile Gly Ile Leu Thr Val (SEQ ID NO: 11)

Glu Ala Met Gly Ile Gly Ile Leu Thr Val (SEQ ID NO: 12)

Tyr Ala Ala Gly Ile Gly Ile Leu Thr Val (SEQ ID NO: 13)

Phe Ala Ala Gly Ile Gly Ile Leu Thr Val (SEQ ID NO: 14)

Ala Ala Ala Gly Ile Gly Ile Leu Thr Val (SEQ ID NO: 15) and

Ala Leu Ala Gly Ile Gly Ile Leu Thr Val (SEQ ID NO: 16)

Three other control peptides were used, i.e.:

Glu Ile Leu Gly Phe Val Phe Thr Leu (SEQ ID NO: 17)

Glu Val Asp Pro Ile Gly His Leu Tyr (SEQ ID NO: 18) and

Phe Leu Trp Gly Pro Arg Ala Leu Val (SEQ ID NO: 19).

SEQ ID NOS: 17 and 18 correspond to amino acids 58-66 of Influenza Amatrix protein (“FLUMA”), and amino acids 168-176 of MAGE-3 TRAP.

In these experiments a peptide known to bind to HLA-A*0201 was used,i.e. amino acids 271-279 of the MAGE-3 TRAP (SEQ ID NO: 19), in aninhibition assay, together with cytolytic T cell line 198NS (Valmori, etal., Canc. Res. 57:735 (1997), which recognizes complexes of SEQ ID NO:19 and HLA-A*0201. In these assays, varying concentrations of testpeptides (1 μM to 100 μM) were incubated with ⁵¹Cr labelled T2 cells(1000 cells/well), for 15 minutes, at room temperature. A suboptimaldose of SEQ ID NO: 19 was then added (1 nM), together with CTL 198NS, inan amount sufficient to create a 5/1 effector/target ratio. A ⁵¹Crrelease assay was then carried out, in accordance with the method setforth, supra. The amount of test peptide needed to inhibit recognitionof complexes by the CTL was calculated, and then binding affinity ofeach peptide, relative to SEQ ID NO: 2, was calculated, using theformula:$R = \frac{{ID}_{50}\text{(SEQ ID NO:2)}}{{ID}_{50}\text{(test peptide)}}$

If R is greater than one, then the tested peptide bound to HLA-A*0201with greater affinity than SEQ ID NO: 2. A value less than one indicateslower affinity. The results are set forth below:

TABLE II Competitor Relative competitor Peptide Sequence [μM] 50%activity Melan-A₂₇₋₃₅ AAGIGILTV 60 1 (SEQ ID NO: 2) ALGIGILTV 1.5 40(SEQ ID NO: 5) AMGIGILTV 2 30 (SEQ ID NO: 6) LAGIGILTV 65 1 (SEQ ID NO:7) MAGIGILTV 55 1 (SEQ ID NO: 8) Melan-A₂₆₋₃₅ EAAGIGILTV 15 4 (SEQ IDNO: 1) ELAGIGILTV 6.5 9 (SEQ ID NO: 9) EMAGIGILTV 20 3 (SEQ ID NO: 10)EALGIGILTV 100 0.6 (SEQ ID NO: 11) EAMGIGILTV 100 0.6 (SEQ ID NO: 12)YAAGIGILTV 4 15 (SEQ ID NO: 13) FAAGIGILTV 2 30 (SEQ ID NO: 14)Influenza A GILGFVFTL 1 60 matrix₅₈₋₆₆ (SEQ ID NO: 17) MAGE-3₁₆₈₋₁₇₆EVDPIGHLY >100 <0.6 (SEQ ID NO: 18)

SEQ ID NOS: 1, 5, 6, 9, 10, 13 and 14 all showed higher affinity thanSEQ ID NO: 2.

EXAMPLE 4

One concern in developing MHC binding peptides is that the resultingcomplexes of MHC molecule and peptide be stable, preferably more stablethan the peptide originally found complexed to the MHC molecule.

To test stability of the newly synthesized peptides, T2 cells wereincubated, overnight, at room temperature in serum free medium withsaturating amounts of peptide (10 uM), and 3 μg/ml β-microglobulin, tofacilitate the assembly of the requisite MHC molecules. Peptides werethen removed, and 10⁻⁴M ementine (which inhibits protein synthesis) wasadded. The cells were then incubated at 37° C. for varying periods oftime. Aliquots of cells were stained, at various points of theincubation, with a labelled HLA-A2 specific mAb to measure HLA-A2expression.

Stability was determined by comparison with complexes involving SEQ IDNO: 17 which were stable over a 6 hour period. The results are presentedin FIGS. 2A-D. FIG. 2A shows mean fluorescence intensity for eachpeptide. “NC” refers to HLA-A*0201, presented by T2 cells, in theabsence of exogenous peptide, while “FLUMA” is SEQ ID NO: 17, and is anacronym for “Flu matrix antigen.” In FIG. 2A, the peptides are SEQ IDNOS: 2, 1 and 9. In FIG. 2B, they are SEQ ID NOS:2, 7, 8, 5, 6 and 17(“FLUMA”). In FIG. 2C, they are SEQ ID NOS: 1, 12, 11, 9, 10 and 17. InFIG. 2D, they are SEQ ID NOS: 1 13, 14 and 17. The breakup is solely tofacilitate the review. FIGS. 2B-2D show relative complex stability wherefluorescent intensities with test peptides were normalized, relative tothe stability observed when using SEQ ID NO:17. SEQ ID NOS:1 and 2 bothform unstable complexes, which decay within one hour. This was alsofound with SEQ ID NOS:7 and 8. On the other hand, SEQ ID NOS: 9, 10, 13and 14 formed stable complexes over a 6 hour period, while SEQ ID NOS:5,6, 11 and 12 formed complexes of intermediate stability.

EXAMPLE 5

The antigenic activity of each of the peptides presented, supra, whenthe peptide was associated with HLA-A*0201, was tested in a ⁵¹Cr assayof the type discussed, supra, using TILNs, and CTLs. Dose responseanalyses were performed on each peptide, and antigenic activity,relative to SEQ ID NO:2, was calculated. These values are set forth inthe following Tables III and IV and FIGS. 3 and 4 which present datafrom TILNs, (Table III and FIG. 3) and CTLs (Table IV and FIG. 4),respectively.

Substituting the N-terminal amino acid of SEQ ID NO: 2 with Leu or Metenhanced activity between 7.5 and 20 fold, while substitutions at thesecond position nearly abolished it, even though binding to HLA-A*-0201was increased (Table III and FIG. 3).

SEQ ID NO:1 was better recognized than SEQ ID NO: 2, and substitution ofAla in the second position of SEQ ID NO:1 increased recognition 30- and600 fold, respectively. Such substitutions at position 3 reducedactivity, which was expected. Substitution of position 1 resulted in anincrease in recognition.

TABLE III TILN LAU 203 TILN LAU 132 Relative Relative Peptide Sequence[nM] activity [nM] activity AAGIGILTV 60 1 30 1 (SEQ ID NO: 2)ALGIGILTV >1000 <0.6 >1000 <0.03 (SEQ ID NO: 5) AMGIGILTV >1000<0.6 >1000 <0.03 (SEQ ID NO: 6) LAGIGILTV 6 10 1.5 20 (SEQ ID NO: 7)MAGIGILTV 8 7.5 2.5 12 (SEQ ID NO: 8) EAAGIGILTV 12 5 3 10 (SEQ IDNO: 1) ELAGIGILTV 2 30 0.05 600 (SEQ ID NO: 9) EMAGIGILTV 2 30 0.05 600(SEQ ID NO: 10) EALGIGILTV >1000 <0.06 >1000 <0.03 (SEQ ID NO: 11)EAMGIGILTV >1000 <0.06 >1000 <0.03 (SEQ ID NO: 12) YAAGIGILTV 5 20 1 30(SEQ ID NO: 13) FAAGIGILTV 1 60 0.05 600 (SEQ ID NO: 14)

The results obtained with CTLs are presented herein. Specifically, fiveindependent HLA-A*0201 restricted Melan-A specific CTL clones were used,each of which is known to lyse melanoma target cells.

The CTLs recognized SEQ ID NO:2 with varying efficiency. When Leu wasused to substitute Ala at position 1, four of the five clones showedenhanced recognition, while similar substitutions at position 2 resultedin a loss of activity. Three of the five clones recognized SEQ ID NO:1more efficiently than SEQ ID NO:2, but all recognized SEQ ID NO:9 veryefficiently, while recognition of SEQ ID NO:10 resulted in decreasedefficiency of recognition to differing degrees, and SEQ ID NO:11resulted in reduced recognition for four of five. When SEQ ID NO:12 wastested, it was surprising that recognition improved, because TILrecognition decreased. With respect to SEQ ID NOS:13 and 14, there wasreduced recognition by the CTLs.

It can be gathered from this that SEQ ID NOS:7 and 9 were betterrecognized, consistently, than the other peptides tested, while otherpeptides were recognized to different degrees.

TABLE IV Recognition of peptide analogs by Melan-A specific CTL clonesRecognition by clone M77.86 7.10 Mel 1.33 M77.80 1.13 SEQ PeptidePeptide Relative Peptide Relative Peptide Relative Peptide RelativePeptide Relative ID NO: sequence [nM] 50% activity [nM] 50% activity[nM] 50% activity [nM] 50% activity [nM] 50% activity 2 AAGIGILTV 15 150 1 300 1 300 1 4000 1 5 ALGIGILTV 90 0.16 >1000 <0.015 >1000<0.3 >1000 <0.3 >10000 <0.4 6 AMGIGILTV >1000 <0.015 >1000 <0.015 >1000<0.3 >1000 <0.3 >10000 <0.4 7 LAGIGILTV 0.08 187 1.5 33 150 2 0.03 1000030 130 8 MAGIGILTV 0.6 .25 15 3 200 1.5 0.5 600 80 50 1 EAAGIGILTV 0.15100 4 12 0.06 5000 600 0.5 2000 2 11 EALGIGILTV 300 0.05 >1000 <0.015 407.5 >1000 <0.3 >10000 <0.4 12 EAMGIGILTV 0.5 30 1 50 0.02 15000 5 60 5080 9 ELAGIGILTV 0.015 1000 0.5 100 0.015 20000 0.5 600 20 200 10EMAGIGILTV 550 36 >1000 <0.015 40 7.5 >1000 <0.3 >10000 <0.4 13YAAGIGILTV 0.015 1000 35 1.4 >1000 <0.3 1000 0.3 >10000 <0.4 14FAAGIGILTV 0.005 3000 7 7 >1000 <0.3 >1000 <0.3 200 20 Relativeantigenic activity of Melan-A derived peptides was measured as describedin the legend to FIG. 4 & table II

EXAMPLE 6

Based upon the preceding data, the peptide of SEQ ID NO:9 was used forCTL induction studies.

In accordance with Valmori, et al, supra, peripheral blood lymphocytesfrom HLA-A*0201 positive melanoma patients were purified bycentrifugation, and were enriched for CD3⁺ cells. The enrichedsubpopulation was then selected for CD8⁺ cells. The resultingsubpopulations routinely contained better than 90% CD8⁺ cells, and thesewere used in further experiments.

The purified, CD8⁺ T cells were plated, at 1−2×10⁶ cells/well, togetherwith 2×10⁶ stimulator cells, the preparation of which is discussed,infra. The effector and stimulator cells were combined in a total of 2ml of Iscove's medium which had been supplemented with 10% human serum,L-arginine (0.55 mM), L-asparagine (0.24 mM), and L-glutamine (1.5 mM),together with recombinant human IL-7 (10 ng/ml) and recombinant humanIL-2 (10 U/ml).

To prepare the stimulator cells, 2×10⁶ autologous PBLs were incubatedfor 2 hours, at 37° C., in serum free medium, with 20 μg/ml of eachpeptide and 3 μg/ml β2-microglobulin. The PBLs were then washed,irradiated, (3000 rads), and then adjusted to an appropriate volume,before being added to the CD8⁺ cell populations. On day 7, cells wererestimulated with peptide pulsed, autologous PBLs in complete medium,supplemented with 10 ng/ml of recombinant human IL-7, and 10 U/ml ofrecombinant human IL-2. There were weekly restimulations, using PBLswhich were peptide pulsed and irradiated. CTL activity was tested forthe first time after the second cycle (MC-2).

The results are shown in the following table and in FIG. 4. In FIG. 4,the source of CD8⁺ cells used was LAU203. CTL activity was assayed sevendays after the second (MC-2) restimulation. Results were obtained usingSEQ ID NOS: 1 and 2. These were used to permit comparison to SEQ ID NO:9.

Note that there was barely any activity with the parental peptides insample LAU203, while SEQ ID NO: 9 provoked a strong CTL response. Thisactivity was also cross reactive with SEQ ID NO: 1.

The results in the following table describe experiments using the samepeptides and using PBL from eight different HLA-A2 positive melanomapatents, LAU203, LAU182, LAU145, LAU86, LAU50, LAU148, LAU161 andLAU119.

Additional experiments are depicted in FIG. 3 which show recognition ofvarious Melan-A peptide analogues presented by T2 cells, by TILN LAU203and TILN LAU132. A four hour ⁵¹Cr assay was conducted at a lymphocyte totarget ration of 30:1.

The first panels of FIG. 3 (top and bottom) compare SEQ ID NOS: 2, 7, 8,5 and 6.

The second set of panels (top and bottom) compare SEQ ID NOS: 1, 9, 10,11 and 12.

The third set (top and bottom) compares SEQ ID NOS: 1, 13, 14 and 4.

The most important result obtained herein, however, was the fact thatCTLS, induced with SEQ ID NO: 9 did recognize and lyse cells presentingthe endogenous peptide when SEQ ID NO: 9 was used.

TABLE V Percentage specific lysis from cultures stimulated withpeptide^(a)): tested on: SEQ ID NO:2 SEQ ID NO:1 SEQ ID NO:9 PatientMelan-A 27-35 Melan-A 26-35 Melan-A 26-35 A27L code E/T^(b) T2 T2 + M10Me260 Me290 T2 T2 + M10 Me290 Me260 T2 T2 + M10 Me290 Me260 LAU203 100 38^(c)) 29 7 17 37 41 15 6 32 83 18 81 30 29 11 10 0 17 23 7 1 26 96 475 10  3  6 2 0 9 17 0 0 17 73 1 62 LAU132 100  9 12 1 0 19 19 6 3 34 506 31 30  3  7 2 0 5 10 1 2 16 32 3 18 10  0  0 5 1 0 0 1 0 5 23 2 6LAU145 100 15 24 4 1 39 40 5 9 29 50 6 30 30  9 12 3 1 15 25 2 1 10 29 519 10  3  6 0 0 4 6 0 0 10 16 3 7 LAU86 100 36 29 22 5 64 38 14 10 35 4524 15 30 17 15 9 5 20 26 6 0 24 23 10 4 10 16  5 2 0 10 10 1 0 14 9 1 0LAU50 100 21 26 7 5 18 20 5 5 19 26 6 20 30  7 16 4 5 8 13 1 0 10 18 3 810  7  7 0 4 0 4 1 0 3 12 0 0 LAU148 100 51 39 13 4 46 45 9 0 34 39 9 430 19  8 5 4 20 26 1 2 19 27 9 3 10  3  6 1 0 14 14 6 0 13 13 1 0 LAU161100 24 22 6 1 33 31 3 1 25 38 4 23 30  3  8 6 1 16 12 3 0 18 23 2 13 10 2  0 5 0 9 7 2 0 5 11 3 4 LAU119 100 31 27 5 12 33 31 1 4 18 46 5 45 30 7 13 1 1 17 23 3 4 13 39 4 25 10  4  0 0 0 9 12 1 0 7 17 2 16 Clone 610  7 78 2 73  3  3 74 0 61  1  0 65 0 51 ^(a))Lytic activity wasassayed 7 days after the third restimulation. ^(b))Lymphocyte to targetcell ratio titration was performed for every assay. ^(c))Numbersrepresent the percent specific lysis obtained for each target. Me290 isa Melan-A and HLA-A*0201 positive melanoma cell line obtained frompatient LAU203. Me260 is a HLA-A*0201 negative melanoma cell lineobtained from patient LAU149. Each number represents the geometric meanof duplicate cultures. Bold face type indicate significant specific CTL.When the differences in specific lysis obtained on T2 cells in presenceor in absence of Melan-A 26-35 (1 μM) or Me290 and Me260 is equal orhigher than 10%. A patient is considered as responder when a significantspecific lysis is detected in at least one of the cultures. ^(d))Clone 6is a Melan-A specific CTL clone derived from the TILN 289.

EXAMPLE 7

As was pointed out, supra, the decapeptide of SEQ ID NO: 1 had a higherefficiency of recognition than the nonamer of SEQ ID NO: 2. Experimentswere carried out to determine if this was the result of better bindingof the peptide of SEQ ID NO: 1 to HLA-A*0201 molecules. These involved afunctional peptide competition assay. This type of assay is described byGaugler et al., J. Exp. Med. 174: 921 (1994), incorporated by reference,but is described herein. In this assay, HLA-A*0201 expressing targetcells (T2 cells) were labelled with ⁵¹Cr and than incubated, for 15minutes, with varying concentrations of peptides. A suboptimumconcentration of mutant Ras 5-14 peptide was added. This peptide hasamino acid sequence:

Lys Leu Val Val Val Gly Ala Val Gly Val (SEQ ID NO: 20).

After 15 minutes a sample of CTL clone 7 RAS was added. This CTL clonehad been obtained from the draining lymph node of an HLA-A*0201human/β-microglobulin double transgenic mouse that had been injectedwith the peptide of SEQ ID NO: 20. The CTLs were added at a ratio of 5lymphocytes (10,000 cells/well): 1 target cell. The cells were incubatedat 37° C. for four hours, and then the assay was terminated. In additionto the peptide of SEQ ID NO: 1, those of SEQ ID NO: 2, SEQ ID NO: 13,SEQ ID NO: 14, SEQ ID NO: 3, SEQ ID NO: 4, and

Glu Ala Asp Pro Thr Gly His Ser Tyr (SEQ ID NO: 21),

were tested. SEQ ID NO: 21 is a known peptide, derived from MAGE-1,which is known to bind to HLA-A1 molecules and stimulate lysis. See U.S.Pat. No. 5,405,940, SEQ ID NO: 12, incorporated by reference.

When SEQ ID NO: 20 was used alone, the lysis percentage was 80%. Controllysis, with no peptide, was 4%.

The results indicated that SEQ ID NO: 1 showed five fold more efficientbinding than SEQ ID NO: 2. Both SEQ ID NOS: 3 and 4 bound withactivities comparable to SEQ ID NO: 2, while the control (SEQ ID NO:21), showed no binding. Both of SEQ ID NOS: 13 and 14 showedsignificantly improved binding as compared to SEQ ID NO: 2. Similarresults were obtained when a human CTL clone, specific for a complex ofHLA-A*0201 and a different peptide, was used. The table which followspresents relative competitor activity as concentration of SEQ ID NO: 2required to inhibit control lysis by 50%, divided by concentration ofpeptide being tested to secure the same result:

TABLE VI Relative Competitor Activity Peptide Exp1 Exp2 Exp3 SEQ ID NO:2 1 1 1 SEQ ID NO: 1 4 4 5  SEQ ID NO: 14 12 17 20  SEQ ID NO: 13 8 1010 SEQ ID NO: 3 Not Done Not Done 2 SEQ ID NO: 4 Not Done Not Done 2

EXAMPLE 8

Relative HLA-A*0201 peptide binding activity was then determined inanother assay, based upon a flow cytometric assay. In these experiments,2×10⁵ T2 cells were incubated with varying concentrations of thepeptides of SEQ ID NO: 1, 2, 13, 14 or 4, for 16 hours, at 23° C., inthe presence of 2 ug/ml of human β₂ microglobulin. Cells were washed, at4° C., and then stained with monoclonal antibody BB7.2, labelled withFITC. This mAb is specific for a conformation dependent epitope onHLA-A2 molecules. Fluorescence index was then calculated, by using theformula (mean fluorescence of sample—mean fluorescence ofbackground)/(mean fluorescence of background). See Nijman et al., Eur.J. Immunol. 23: 1215 (1993). Again, SEQ ID NO: 1 showed highestefficiency of binding (about 10 fold) than SEQ ID NO: 2. SEQ ID NO: 4showed relative binding activity about the same as SEQ ID NO: 2, whileSEQ ID NOS: 13 and 14 had binding activities comparable to SEQ ID NO: 1.

EXAMPLE 9

In these experiments, efficiency of peptide recognition was assessedwith a panel of 13 CTL clones, all of which were specific for complexesof HLA-A*0201 and SEQ ID NO: 2.

The same type of ⁵¹Cr release assay as is described in, e.g., example 3,supra was carried out. Specifically, T2 cells were labelled with ⁵¹Cr(the sodium salt was used), in Tris-Dulbecco buffer, supplemented with 2mg/ml bovine serum albumin, and a 1:40 dilution of W6/32 ascites asdescribed by Gaugler et al, supra. This stabilizes the MHC molecule. Thelabelled cells were added to varying concentrations of peptides, and invarying amounts so as to create differing effector:target ratios. Ten ofthe thirteen CTL clones tested recognized the decapeptide of SEQ ID NO:1 more efficiently than the nonapeptide of SEQ ID NO: 2, requiringconcentrations of anywhere from 20 to more than 1000 fold lower than thenonapeptide to achieve half-maximal lysis. The three remaining CTLsyielded titration curves which were similar. None of the CTLs recognizedcomplexes of SEQ ID NO: 3 and HLA-A2. In additional experiments, one ofthe CTL clones was tested in IL-2 release assays, with SEQ ID NO: 1again proving to be I0.fold more efficient than SEQ ID NO: 2.

EXAMPLE 10

SEQ ID NOS: 1, 13 and 14 were then used to “dissect” the panel of theCTLs described supra. A recognition assay of the type described inexample 7, supra, was carried out using these peptides. Four of the CTLsrecognized SEQ ID NOS: 1, 13 and 14 equally well. A fifth CTL recognizedSEQ ID NOS: 1 and 13, but not SEQ ID NO: 14. Two other CTLs recognizedSEQ ID NOS: 1 and 14, but not 13. One CTL only recognized SEQ ID NO: 1.

EXAMPLE 11

A set of experiments were then carried out to study the receptors of theT cells described herein (“TCRs” hereafter), because it is known thatdifferent elements combine in the TCR repertoire, forming different TCRsas a result.

To do this, total RNA of 10⁶ cells of each CTL clone tested wasextracted, following Chomczynski et al., Anal. Biochem. 162: 156 (1987).Then, reverse transcription using a poly(dT) primer was carried out,following the instructions in a commercially available product.Following this, aliquots of samples were PCR amplified, using a panel ofVα and Vβ probes, and Cα/Cβ specific oligonucleotides, in accordancewith Genevee, et al., Eur. J. Immunol. 22: 1261 (1992), incorporated byreference. Six different Vα segments were found, i.e., Vα2, 4, 6, 7, 14and 21. One clone actually presented two in-frame Vα transcripts. Sevendifferent Vβ segments were found (two clones expressed Vβ13, twoexpressed Vβ14, and two expressed Vβ16. Vβ2, Vβ3, Vβ7.2 and Vβ38.2 wereeach expressed by one clone).

EXAMPLE 12

The determination of the contribution of single amino acid side chainsto the interaction between SEQ ID NO: 1 and HLA-A*0201 molecule, wasstudied, by testing single Ala substituted derivatives. That is to say,derivatives were prepared which were identical to SEQ ID NO: 1 but for achange at position 1, 4, 5, 6, 7, 8, 9 or 10 to Ala.

The peptides were prepared, following standard synthetic methods. Then,they were tested in a functional competition assays based upon theirability to inhibit binding of a known HLA-A*0201 binding peptide, i.e.

Tyr Met Asp Gly Thr Met Ser Gln Val (SEQ ID NO: 22),

derived from tyrosinase, and an HLA-A*0201 restricted CTL clone, LAU132/2. In brief, T2 cells were labelled with ⁵¹Cr in the presence ofmonoclonal antibody W6/32. Both the cells and monoclonal antibody aredescribed supra. Varying concentrations of competitor peptide (50 ulvolumes) were incubated with 50 ul of the ⁵¹Cr labelled T2 cells (Thisquantity gives 1000 cells/well) for 15 minutes, at room temperature.Then, 1 nM of the peptide of SEQ ID NO: 22 was added, which is asuboptimal dose, together with 5000 CTLs, per well (a volume of 50 ul).⁵¹Cr release was measured after incubating for four hours at 37° C. Theconcentration of each competitor peptide required to inhibit ⁵¹Crrelease by 50% was determined. Comparison was facilitated by calculatingrelative competitor activity as the concentration of SEQ ID NO: 1 neededfor 50% inhibition, divided by the 50% inhibition value for the testpeptide.

It was found that substituting Ala for Glu at position 1 (SEQ ID NO: 15)resulted in a 5 fold increase in competitor activity. Substitution ofGlu at position 4 or 6 by Ala resulted in decreased activity, of 20 and10 fold, as did substitution of positions 7 and 10 (about 25 fold, eachtime). Changes at positions 8 or 9, i.e.

Glu Ala Ala Gly Ile Gly Ile Ala Thr Val (SEQ ID NO: 23)

and

Glu Ala Ala Gly Ile Gly Ile Leu Ala Val (SEQ ID NO: 24)

did not result in significant changes in activity.

EXAMPLE 13

The stability of complexes formed by the single Ala substitutions of SEQID NO: 1, discussed supra, and HLA-A 0201 was then studied. Briefly, T2cells were loaded with saturating concentrations (10 uM) of the analogs,and incubated overnight at room temperature with the peptide and β2microglobulin (3 ug/ml), in serum free medium. Excess peptide wasremoved, and ementine (10⁻⁴M) was added to block protein synthesis.Cells were then incubated, for varying time periods, and aliquots werestained with fluorescent labelled anti HLA-A2 monoclonal antibody(BB7.2), to determine the amount of the molecules on the surface. Sincethe peptide of SEQ ID NO: 17, supra, is known to form stable complexeswith HLA-A*0201 (van der Bruggen, J. Immunol. 156: 3308 (1996)), thispeptide was used as an internal standard. Complex stability wasdetermined by calculating mean fluorescence with analog, subtractingbackground fluorescence and dividing by the same values obtained usingSEQ ID NO: 17. Background fluorescence was the value obtained using T2cells treated similarly, but without peptide.

It was found that, over a period of 1-6 hours, complexes of SEQ ID NO:1/HLA-A*0201 were unstable, and dissociated in less than an hour. SEQ IDNO: 15 formed stable complexes over the same six hour period. All otherderivatives tested formed complexes with low stability.

EXAMPLE 14

The derivatives described supra were then tested for their relativeantigenic activity. In these experiments, two TILN populations, i.e.,TILN LAU 132 and TILN LAU 203, preparation of which is described, supra,and a panel of ten different cytolytic T cell lines were tested. Of theten CTLs, five were derived from infiltrating lymphocytes or tumorinfiltrating lymph nodes, and five were from normal donor peripheralblood lymphocytes. All were known to be specific for complexes ofHLA-A*0201 and SEQ ID NO: 2; however, given the results discussed supra,showing superiority of SEQ ID NO: 1, this decapeptide was used forcomparison.

Antigen recognition was assessed in a ⁵¹Cr release assay. Target, T2cells were labelled with ⁵¹Cr for one hour at 37° C., then washed,twice. The labelled target cells (1000 cell samples in 50 ul) were thenincubated with varying concentrations of peptides (in 50 ul volume), for15 minutes at room temperature, before adding effector cells (50 ul).When TILNs were the effector cells, these had been preincubated for atleast 20 minutes at 37° C., with unlabelled K562 cells (50,000cells/well), to eliminate non-specific lysis due to NK-like effectors.The ⁵¹Cr was measured in supernatant, harvested after four hours ofincubation at 37° C. Percent lysis was determined by subtractingspontaneously released ⁵¹Cr from ⁵¹Cr released with the tested, dividedby a figure obtained by subtracting spontaneous release from total ⁵¹Cr,and multiplying the resulting figure by 100. Titration was carried outover concentrations ranging from 10⁻⁵ to 10⁻¹³M. For quantitativecomparison, concentrations required for 50% maximal activity, normalizedagainst reference values for SEQ ID NO: 2 were determined.

SEQ ID NO: 15 was found to be recognized 20-60 fold better than theparental decapeptide of SEQ ID NO: 1, by the two TILN populations, incontrast to the other variants tested. With respect to the CTLs, 8 of 10of those tested recognized the peptide better than they recognized SEQID NO: 1 or 2.

Additional differences were observed with respect to the CTLspecificity. Five of the ten CTLs tested recognized SEQ ID NO: 1 betterthan SEQ ID NO: 2. One of these five CTLs recognized SEQ ID NO: 1efficiently, and SEQ ID NO: 2 poorly. Three of the remaining five clonesrecognized SEQ ID NOS: 1 and 2 equally efficiently, and two recognizedSEQ ID NO: 2 better than SEQ ID NO: 1.

What this shows is a strong degree of diversity in fine specificity oftumor reactive CTLs.

EXAMPLE 15

A further set of experiments were then carried out to determine ifdiversity of antigenic specificity, as displayed by the CTL clones,supra, was a general characteristic of the T cell repertoire for theantigen SEQ ID NO: 1 or was attributable to different methodologies usedto derive the CTLs. To test this, the source of the specific T cells hadto be one which had not been subjected to antigen driven selection fromin vitro stimulation with peptides. The TILN population LAU 203,described supra, was used. As was shown in the prior examples, thepopulation exhibits relatively high CTL activity against Melan-AHLA-A*0201 positive melanoma cells. CTL clones were derived from thispopulation, by limiting dilution cultures, in the presence ofirradiated, allogenic PBMCs, Epstein Barr Virus transformed Blymphocytes, phytohemagglutinin, and recombinant IL-2. Using standardprobability models, clones were derived from cultures having probableclonality of higher than 90%. These were then expanded, by plating 5×10³cells, every 3-4 weeks, into microtiter plates, together with irradiatedfeeder cells (5×10⁴ allogenic PBMCs, and 2.10⁴ EBV transformed B cells),with PHA and recombinant IL-2.

Two independent experiments were carried out, resulting in 130 growingclones. When these were tested in a ⁵¹Cr release assay, of the typedescribed supra, 11 of these were found to recognize at least one of SEQID NO: 1 and 2.

In order to determine fine specificity, the type of antigen recognitionassay described supra was carried out, using SEQ ID NOS: 1, 2 and 15. Itwas found that four clones recognized SEQ ID NO: 1 better than SEQ IDNO: 2 (i.e., relative antigenic activity was at least 10 fold greater),six clones recognized the two peptides equally well, and one recognizedSEQ ID NO: 2 better than SEQ ID NO: 1. Nine CTLs recognized SEQ ID NO:15 better than SEQ ID NO: 1 and 2, with one of the clones actuallyrecognizing SEQ ID NO: 15 at nanomolar concentrations, in contrast tomicromolar concentrations for the peptides SEQ ID NO: 1 and SEQ ID NO:2.

The foregoing examples, as will be seen, describe the various featuresof the invention. These include peptides which bind to HLA molecules,such as HLA-A2 molecules, exemplified by HLA-A*0201, which may alsoprovoke proliferation of cytolytic T cells, These peptides, as will beseen from the data herein, are nonapeptides or decapeptides. As with allpeptides, the first amino acid is the amino terminus, and the last oneis the carboxy terminus. The peptides of the invention may bedecapeptides, which have a Val moiety at the C, or carboxy terminus.They may have at the amino terminus, Tyr or Phe when the second aminoacid is Ala. In another embodiment, the amino terminus is Glu followedby Ala, Leu or Met in the second and third position, and terminate withVal, wherein if position two is Ala, position three must be Met or Leu,and vice versa. The peptides having the amino acid sequences set forthin any of SEQ ID NOS: 5, and 8-14 are exemplary.

Also a part of the invention are isolated cytolytic T cell lines whichare specific for complexes of these peptides and their MHC bindingpartner, i.e., an HLA molecule, such as an HLA-A2 molecule, HLA-A*0201being especially preferred.

The ability of these peptides to bind to HLA molecules makes them usefulas agents for determining presence of HLA-A2 positive cells, such asHLA-A*0201 positive cells, by determining whether or not the peptidesbind to cells in a sample. This “ligand/receptor” type of reaction iswell known in the art, and various methodologies are available fordetermining it.

A further aspect of the invention are so-called “mini genes” which carryinformation necessary to direct synthesis of modified decapeptides viacells into which the mini genes are transfected. Mini genes can bedesigned which encode one or more antigenic peptides, and are thentransferred to host cell genomes via transfection with plasmids, or viacloning into vaccinia or adenoviruses. See, e.g., Zajac, et al., Int. J.Cancer 71: 496 (1997), incorporated by reference.

The peptides may be combined with peptides from other tumor rejectionantigens to form ‘polytopes’. Exemplary peptides include those listed inU.S. patent application Ser. Nos. 08/672,351, 08/718,964, now U.S. Pat.No. 5,932,694, respectively, Ser. No. 08/487,135 now U.S. Pat. No.5,821,122, Ser. No. 08/530,569 now U.S. Pat. No. 5,939,526, and Ser. No.08/880,963, now U.S. Pat. No. 6,025,470, all of which are incorporatedby reference.

Additional peptides which can be used are those described in thefollowing references, all of which are incorporated by reference: U.S.Pat. Nos. 5,405,940; 5,487,974; 5,519,117; 5,530,096; 5,554,506;5,554,724; 5,558,995; 5,585,461; 5,589,334; 5,648,226; and 5,683,886;PCT International Publication Nos. 92/20356; 94/14459; 96/10577;96/21673; 97/10837; 97/26535; and 97/31017 as well as U.S. applicationSer. No. 08/713,354, now abandoned.

Polytopes are groups of two or more potentially immunogenic or immunestimulating peptides, which can be joined together in various ways, todetermine if this type of molecule will stimulate and/or provoke animmune response.

These peptides can be joined together directly, or via the use offlanking sequences. See Thompson et al. Proc. Natl. Acad. Sci. USA92(13): 5845-5849 (1995), teaching the direct linkage of relevantepitopic sequences. The use of polytopes as vaccines is well known. See,e.g. Gilbert et al., Nat. Biotechnol. 15(12): 1280-1284 (1997); Thomsonet al., supra; Thomson et al., J. Immunol. 157(2) 822-826 (1996); Tam etal., J. Exp. Med. 171(1): 299-306 (1990), all of which are incorporatedby reference. The Tam reference in particular shows that polytopes, whenused in a mouse model, are useful in generating both antibody andprotective immunity. Further, the reference shows that the polytopes,when digested, yield peptides which can be and are presented by MHCs.Tam shows this by showing recognition of individual epitopes processedfrom polytope ‘strings’ via CTLs. This approach can be used, e.g., indetermining how many epitopes can be joined in a polytope and stillprovoke recognition and also to determine the efficacy of differentcombinations of epitopes. Different combinations may be ‘tailor-made’for the patients expressing particular subsets of tumor rejectionantigens. These polytopes can be introduced as polypeptide structures,or via the use of nucleic acid delivery systems. To elaborate, the arthas many different ways available to introduce DNA encoding anindividual epitope, or a polytope such as is discussed supra. See, e.g.,Allsopp et al., Eur. J. Immunol. 26(8); 1951-1959 (1996), incorporatedby reference. Adenovirus, pox-virus, Ty-virus like particles, plasmids,bacteria, etc., can be used. One can test these systems in mouse modelsto determine which system seems most appropriate for a given, parallelsituation in humans. They can also be tested in human clinical trials.

Also a feature of the invention is the use of these peptides todetermine the presence of cytolytic T cells in a sample. It was shown,supra, that CTLs in a sample will react with peptide/MHC complexes.Hence, if one knows that CTLs are in a sample, HLA-A2 positive cells canbe “lysed” by adding the peptides of the invention to HLA-A2 positivecells, such as HLA-A*0201 positive cells, and then determining, e.g.,radioactive chromium release, TNF production, etc. or any other of themethods by which T cell activity is determined. Similarly, one candetermine whether or not specific tumor infiltrating lymphocytes(“TILs”) are present in a sample, by adding one of the claimed peptideswith HLA-A2 positive cells to a sample, and determining lysis of theHLA-A2 positive cells via, e.g., ⁵¹Cr release, TNF presence and soforth. In addition, CTL may be detected by ELISPOT analysis. See forexample Schmittel et al (1997). J. Immunol. Methods 210: 167-174 andLalvani et al (1997). J. Exp. Med. 126: 859 or by FACS analysis offluorogenic tetramer complexes of MHC Class I/peptide (Dunbar et al(1998), Currrent Biology 8: 413-416. All are incorporated by reference.

Of course, the peptides may also be used to provoke production of CTLs.As was shown, supra, CTL precursors develop into CTLs when confrontedwith appropriate complexes. By causing such a “confrontation” as itwere, one may generate CTLs. This is useful in an in vivo context, aswell as ex vivo, for generating such CTLs.

Other features of the invention will be clear to the skilled artisan,and need not be repeated here.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

24 1 10 PRT Artificial sequence Peptide based on Melan-A peptide 1 GluAla Ala Gly Ile Gly Ile Leu Thr Val 1 5 10 2 9 PRT Artificial sequencePeptide derived from Melan A 2 Ala Ala Gly Ile Gly Ile Leu Thr Val 1 5 310 PRT Artificial Sequence Peptide based on Melan A protein 3 Ala AlaGly Ile Gly Ile Leu Thr Val Ile 1 5 10 4 9 PRT Artificial SequencePeptide based on Melan A protein 4 Ile Leu Thr Val Ile Leu Gly Val Leu 15 5 9 PRT Artificial Sequence Derivative of SEQ ID NO 2 5 Ala Leu GlyIle Gly Ile Leu Thr Val 1 5 6 9 PRT Artificial Sequence Derivative ofSEQ ID NO 2 6 Ala Met Gly Ile Gly Ile Leu Thr Val 1 5 7 9 PRT Artificialsequence Derivative of SEQ ID NO 2 7 Leu Ala Gly Ile Gly Ile Leu Thr Val1 5 8 9 PRT Artificial Sequence Derivative of SEQ ID NO 2 8 Met Ala GlyIle Gly Ile Leu Thr Val 1 5 9 10 PRT Artificial Sequence Derivative ofSEQ ID NO 1 9 Glu Leu Ala Gly Ile Gly Ile Leu Thr Val 1 5 10 10 10 PRTArtificial sequence Derivative of SEQ ID NO 1 10 Glu Met Ala Gly Ile GlyIle Leu Thr Val 1 5 10 11 10 PRT Artificial sequence Derivative of SEQID NO 1 11 Glu Ala Leu Gly Ile Gly Ile Leu Thr Val 1 5 10 12 10 PRTArtificial Sequence Derivative of SEQ ID NO 1 12 Glu Ala Met Gly Ile GlyIle Leu Thr Val 1 5 10 13 10 PRT Artificial sequence Derivative of SEQID NO 1 13 Tyr Ala Ala Gly Ile Gly Ile Leu Thr Val 1 5 10 14 10 PRTArtificial Sequence Derivative of SEQ ID NO 1 14 Phe Ala Ala Gly Ile GlyIle Leu Thr Val 1 5 10 15 10 PRT Artificial sequence Derivative of SEQID NO 1 15 Ala Ala Ala Gly Ile Gly Ile Leu Thr Val 1 5 10 16 10 PRTArtificial Sequence Derivative of SEQ ID NO 1 16 Ala Leu Ala Gly Ile GlyIle Leu Thr Val 1 5 10 17 9 PRT Artificial sequence Portion of InfluenzaA matrix protein 17 Glu Ile Leu Gly Phe Val Phe Thr Leu 1 5 18 9 PRTArtificial Sequence Peptide from protein MAGE-3 (amino acids 168-176) 18Glu Val Asp Pro Ile Gly His Leu Tyr 1 5 19 9 PRT Artificial SequencePeptide from protein MAGE-3 (amino acids 271-279) 19 Phe Leu Trp Gly ProArg Ala Leu Val 1 5 20 10 PRT Artificial Sequence Mutant Ras 5-14Peptide 20 Lys Leu Val Val Val Gly Ala Val Gly Val 1 5 10 21 9 PRTArtificial Sequence Peptide from MAGE-1 Protein 21 Glu Ala Asp Pro ThrGly His Ser Tyr 1 5 22 9 PRT Artificial Sequence Peptide derived fromtyrosinase which binds HLA-A*0201 22 Tyr Met Asp Gly Thr Met Ser Gln Val1 5 23 10 PRT Artificial Sequence Derivative of SEQ ID NO 15 23 Glu AlaAla Gly Ile Gly Ile Ala Thr Val 1 5 10 24 10 PRT Artificial SequenceDerivative of SEQ ID NO 15 24 Glu Ala Ala Gly Ile Gly Ile Leu Ala Val 15 10

What is claimed is:
 1. An isolated peptide which binds to an HLA-A2molecule and provokes proliferation of cytolytic T cells, said peptideconsisting of ten amino acids, wherein the carboxy terminal amino acidis Val, the amino terminal amino acid is Tyr or Phe, and the secondamino acid is Ala.
 2. An isolated peptide which binds to an HLA-A2molecule and provokes proliferation of cytolytic T cells, wherein saidpeptide has an amino acid sequence selected from the group consistingof, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.